We seek to understand the regulatory mechanisms that control the movements of mitochondria in cells. Mitochondria move and undergo fission and fusion in all eukaryotic cells, but the need to supply mitochondria to the far-flung extremities of neurons creates a particular urgency for mitochondrial transport in neurons. Defects in mitochondrial movements can give rise to peripheral neuropathies and degeneration 1. We and our colleagues have identified a protein complex that includes milton, Miro, and the kinesin-1 heavy chain (KHC) and have shown that this complex is essential for the anterograde ((+)-end directed) transport of mitochondria in neurons. We have determined that Miro is degraded by the proteasome via PINK1/Parkin- dependent phosphorylation and ubiquitination. Additionally, and most relevantly to this proposal, we have obtained exciting preliminary results demonstrating a novel machinery spanning both the mitochondrial outer and inner membranes that stabilizes Miro. We now propose to further our understanding of this machinery by examining in greater detail how it is regulated by intracellular signals and neuronal activity. These signaling pathways are crucial to the ability of mitochondria to properly distribute themselves within a cell and to permit cells to respond to stresses and changes in activity. Aim 1: To determine the nature of the ternary complex spanning the mitochondrial membranes. Aim 2: To determine the regulatory signals of this ternary complex. Aim 3: To determine the functional relevance of this ternary complex.